Cell activation is associated with rapid upregulation of synthesis of phospholipids (PL) that includes phosphatidic acid (PA), diacylglycerol (DAG) and phosphatidylinositol (PI). PA's are a molecularly diverse group of phospholipid second messengers coupled to cellular activation and mitogenesis. Singer et al., Exp. Opin. Invest. Drugs 3:631643, 1994. Compounds capable of modulating PA generation and hence altering a signal involved in cell activation may therefore be of therapeutic interest in the area of inflammation and oncology.
Lysophosphatidic acid acyltransferase (LPAAT) is an important enzyme in the synthesis of a specific species of PA in activated monocytic cells. Rice et al., Proc. Nat'l Acad. Sci. USA 91:3857-3861, 1994. PCPLD is another major enzyme class involved in the generation of PA through hydrolysis of phosphatidyl choline (PC) into PA and choline. Exton, Biochim Biophys Acta 1212:2642, 1994). Okamura et al., J. Biol. Chem. 269:31207-31213, 1994, report PCPLD protein purification from pig lung. Brown et al., J. Biol. Chem. 270:14935-14943, 1995, report PCPLD protein purification from porcine brain, and Vinggaard et al. discuss PCPLD isolation from human placenta. Biochim Biophys Acta 1258:169-176, 1995.
In plant species, Wang et al. published results of cloning efforts with castor bean PCPLDs. J. Biol. Chem. 269:20312-20317, 1994. Ueki et al. disclose PCPLD purified from rice and maize, Plant Cell Physiol. 36:903-914, 1995, and there also are reports on PCPLD isolation and purification from yeast. Ella et al., Biochem. J. 314, 15-19, 1996; Rose et al., Proc. Natl. Acad. Sci. 92: 12151-12155, 1995.
Most recently, Hammond et al. report cloning of a human isoform of PCPLD, hPLD1. J. Biol. Chem. 270: 29640-29643, 1995. SEQ ID NO. 3 is a sequence listing of the amino acids of hPLD1. Based on a variety of biochemical studies including differential subcellular fractionation, distinct mechanism of activation, substrate specificity and different chromatographic properties, evidence for the existence of multiple phospholipase D (PLD) isoforms in mammalian cells is growing rapidly. Liscovitch et al., Chem. Phys. Lipids 80: 37-44, 1996; Kiss, Chem. Phys. Lipids 80: 81-102. hPLD1 has approximately a 40% sequence homology with hPCPLD.
Although other mammalian PLD sequences have been cloned, heretofore the sequence of the disclosed PCPLD has not been obtained. Therefore, cloning cDNA isoforms of PLD that are closely related to other mammalian and plant isoforms of PLD would be useful in conducting discovery research to identify specific agents capable of modulating this enzyme.